PALM3在SIGIRR调控TLR4信号通路中的作用和机制研究

Excessive,or inappropriate activation of TLRs may lead to severe inflammation and immunity-related tissue damage, such as acute lung injury (ALI). To avoid detrimental and severe inflammatory response, single Ig IL-1R－related molecule (SIGIRR) is required for negatively regulating TLRs signaling in the immune system. However,the underlying mechanism remains unclear. Our preliminary study has verified that paralemmin-3(PALM3) is a novel binding protein of SIGIRR by a yeast two-hybrid screen and co-immunoprecipitation(Co-IP) assay. However the down-regulation of PALM3 reduced the inflammatory response in TLR4 signaling and increased the expression of SIGIRR on cell membrane.So we postulate that the combination of PALM3 and SIGIRR inhibit the role of SIGIRR in TLR4 signaling. The goal of the study is to confirm the specific interaction between PALM3 and SIGIRR by GST Pull-down assay, the role of PALM3 on dynamic changes in the subcellular distribution of SIGIRR will be detected by using confocal fluorescent microscope.Moreover, the binding site will be analyzed after co-transfection with wild or mutant of SIGIRR and PALM3 plasmids. Furthermore, the PALM3 mutants will be selected which can bind SIGIRR but not inhibit the negative effect of SIGIRR on TLR4 signal pathway.The PALM3 mutants will also be designed to intervene the process of ALI by interrupting the combination of PALM3 and SIGIRR. If successful, our results will contribute to elucidate the underlying mechanism for regulation of SIGIRR on TLRs and lead to develop the strategy to "hard to treat"inflammatory disease especially ALI.

过激的TLRs炎症反应是导致急性肺损伤（ALI）等疾病的重要原因。SIGIRR是TLRs信号通路中重要的负性调节蛋白，其调控机制不详。本课题组前期用酵母双杂交"钓取"并经免疫共沉淀证实Paralemmin-3（PALM3）是SIGIRR新的结合蛋白；下调PALM3表达可抑制TLR4的炎症反应，并上调细胞膜上SIGIRR的表达，推测PALM3与SIGIRR结合抑制了SIGIRR的负性调节作用。为进一步阐明其作用机制，本项目拟通过荧光共定位、基因重组等方法研究PALM3对SIGIRR细胞亚定位的影响；通过构建突变体等鉴定两蛋白的结合位点；进一步筛选与PALM3竞争结合SIGIRR的突变体蛋白，通过复制ALI小鼠模型，验证封闭PALM3与SIGIRR结合位点治疗ALI效果。本课题从分子、细胞和动物水平阐明PALM3在SIGIRR调控TLR4信号中的作用和机制，为ALI的治疗提供新的靶点和方向。

过激的TLRs炎症反应是导致急性肺损伤（ALI）等疾病的重要原因。SIGIRR是TLRs信号通路中重要的负性调节蛋白，其调控机制不详。本课题组前期用酵母双杂交"钓取"并经免疫共沉淀证实Paralemmin-3（PALM3）是SIGIRR新的结合蛋白；下调PALM3表达可抑制TLR4的炎症反应，并上调细胞膜上SIGIRR的表达，推测PALM3与SIGIRR结合抑制了SIGIRR的负性调节作用。为进一步阐明其作用机制，本项目采用GST-pull down的方法证实PALM3和SIGIRR存在直接相互作用。采用激光共聚焦方法证实PALM3与SIGIRR存在共定位（使用荧光标记抗体PALM3和SIGIRR对小鼠肺组织进行激光共聚焦检测，红色荧光代表PALM3染色，绿色荧光代表SIGIRR染色，黄色荧光代表PALM3和SIGIRR共定位情况，结果黄色荧光在细胞膜上表达）。将PALM3和SIGIRR共转染细胞，与pDsRed2-N1/PALM3组相比，pEGFP-N2/SIGIRR和pDsRed2-N1/PALM3共转染组中，LPS对A549细胞中炎症因子IL-6的刺激作用减弱，表明PALM3可能通过减弱SIGIRR对TLR4介导的炎症因子的负性调控作用；证实PALM3与SIGIRR结合对炎症因子的影响，从而进一步证实PALM3与SIGIRR在TLR4信号通路的负性调控作用。与pDsRed2-N1+LPS组相比，pDsRed2-N1/PALM3+LPS组中SIGIRR在细胞膜蛋白中的表达明显降低，表明PALM3通过影响SIGIRR膜表达介导TLR4对ARDS的调控作用，阐明了PALM3对SIGIRR亚细胞定位的影响，从而进一步揭示了PALM3与SIGIRR结合抑制SIGIRR负性调节作用的机制。与siRNA-C组小鼠比较， siRNA-PALM3小鼠组中炎症因子IL6、IL-1β、TNFα和MIP-2的表达明显下调；肺部炎症有所减轻；BALF中蛋白含量有所减少；肺组织湿干有所减小，死亡率降低。表明封闭PALM3与SIGIRR结合位点对ALI小鼠的治疗作用。本实验从分子、细胞和动物水平阐明了PALM3在SIGIRR调控TLR4信号中的作用和机制，为ALI的治疗提供新的靶点和方向。